Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2 , was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKβ1 -/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

FN3K (fructosamine 3-kinase) is a mammalian enzyme that catalyses the phosphorylation of fructosamines, which thereby becomes unstable and detaches from proteins. The homologous mammalian enzyme, FN3K-RP (FN3K-related protein), does not phosphorylate fructosamines but ribulosamines, which are probably formed through a spontaneous reaction of amines with ribose 5-phosphate, an intermediate of the pentose–phosphate pathway and the Calvin cycle. We show in the present study that spinach leaf extracts display a substantial ribulosamine kinase activity (approx. 700 times higher than the specific activity of FN3K in erythrocytes). The ribulosamine kinase was purified approx. 400 times and shown to phosphorylate ribulose-ε-lysine, protein-bound ribulosamines and also, with higher affinity, erythrulose-ε-lysine and protein-bound erythrulosamines. Evidence is presented for the fact that the third carbon of the sugar portion is phosphorylated by this enzyme and that this leads to the formation of unstable compounds decomposing with half-lives of approx. 30 min at 37 °C (ribulosamine 3-phosphates) and 5 min at 30 °C (erythrulosamine 3-phosphates). This decomposition results in the formation of a 2-oxo-3-deoxyaldose and inorganic phosphate, with regeneration of the free amino group. The Arabidopsis thaliana homologue of FN3K/FN3K-RP was overexpressed in Escherichia coli and shown to have properties similar to those of the enzyme purified from spinach leaves. These results indicate that the plant FN3K/FN3K-RP homologue, which appears to be targeted to the chloroplast in many species, is a ribulosamine/erythrulosamine 3-kinase. This enzyme may participate in a protein deglycation process removing Amadori products derived from ribose 5-phosphate and erythrose 4-phosphate, two Calvin cycle intermediates that are potent glycating agents.

Intact human erythrocytes catalyse the conversion of fructose into fructose 3-phosphate with an apparent K m of 30mM [Petersen, Kappler, Szwergold and Brown (1992) Biochem. J. 284 , 363Ő366]. The physiological significance of this process is still unknown. In the present study we report that the formation of fructose 3-phosphate from 50mM fructose in intact erythrocytes is inhibited by 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine, with an apparent K i of 100 µ M. 31 P NMR analysis of cell extracts incubated with DMF indicated the presence of an additional phosphorylated compound, which was partially purified and shown to be DMF 3-phosphate by tandem MS. Radiolabelled DMF was phosphorylated by intact erythrocytes with an apparent K m (≈ 100 µ M) approx. 300-fold lower than the value reported for fructose phosphorylation on its third carbon. These results indicate that the physiological function of the enzyme that is able to convert fructose into fructose 3-phosphate in intact erythrocytes is probably to phosphorylate fructosamines. This suggests that fructosamines, which are produced non-enzymically from glucose and amino compounds, may be metabolized in human erythrocytes.